The present invention relates to a process for producing microbial copolyesters, particularly microbial hydroxyalkanoate copolymers, from sucrose-containing feedstocks.
Polyhydroxyalkanoates (PHAs) are a family of biopolyesters synthesized and accumulated in bacterial cells as carbon and energy storage. The biopolyesters can be melted and molded like conventional plastics, but completely biodegraded in the environment. Since PHA bioplastics are produced from renewable feedstocks, their fossil energy consumption and greenhouse gas emissions are much lower than those of petroleum-based counterparts.
Poly-(3-hydroxybutyrate), usually identified as P3HB or PHB, is the most common PHA formed by microorganisms from carbohydrates, and can be represented by the following formula:
Formation of other PHAs often needs precursors or structurally related substrates. The hydroxyalkanoate monomers generated via metabolism in native microbial species are usually 3-hydroxyalkanoates (3HAs) in R configuration due to the stereo-specificity of enzymes in PHA biosynthesis.
Because of high stereo-regularity, the biopolyesters exhibit some unique properties such as optical activity, biodegradability, biocompatibility, and high crystallinity of some PHA polymers. For instance, P3HB has a high crystallinity of up to 70%, resulting in a rigid material with high melting temperature (180° C.), high elastic modulus (3.5 GPa), high tensile strength (43 MPa), but low elongation at break (5%). It therefore has limited applications.
The ductility of P3HB can be improved by introducing large side groups, such as ethyl groups, onto the polyester backbone, to form a co-polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), usually identified as P3HB3HV or PHBV, having formula:
Compared to PHB, a PHBV co-polyester (HB:HV=90:10) has a lower crystallinity (60%), lower melting point (140° C.), lower elastic modulus (0.8 G Pa), lower tensile strength (20 M Pa), but higher elongation at break (50%) The 3HV monomers, however, can be incorporated into the crystal lattice of P3HB, a phenomenon called isodimorphism which reduces the function of ethyl group (see e.g. the article by P. J. Barham, P. Barker, S. J. Organ, FEMS Microbiol. Lett. 103: 289-298 (1992)).
By incorporating a long monomer, such as 4-hydroxybutyrate (4HB) into the PHA backbone, crystallinity can also be reduced, and hence properties of the material can be modified. For instance, a co-polyester, P3HB4HB (3HB:4HB=84:16), having formula:
has a crystallinity of 45%, tensile strength of 26 MPa, and elongation at break of 400% (see the article by Z. Zhu, P. Dakwa, P. Tapadia, R. W. Whitehouse, S. Q. Wang, Macromolecules 36: 4891-4897. (2003)). The long monomer seems quite efficient in improving the ductility of co-polyesters. A high content of 4HB, however, results in low tensile strength and slow crystallization.
A ter-polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) (3HB:3HV:4HV=0.8:69.2:30)—usually identified as P3HB3HV4HV or PHBVV—having formula:
was synthesized from 4-ketovaleric acid by using the bacterium P. putida GPp1O4. The ter-polyester is solidified very slowly from melt, which may pose a great challenge to thermal processing and fabrication of the bioplastic (see the article by V. Gorenflo, G. Schmack, R. Vogel and A. Steinbuchel, Biomacromolecules, 2: 45-57 (2001)).
In microbial PHA biosynthesis, the monomers of 3HV, 4HB and 4HV are often derived from precursors or structural related chemicals such as propionic acid, 1,4-butanediol and 4-ketovaleric acid, either as a sole carbon source or a co-substrate with common carbon sources such as glucose. The precursors, however, are often much more expensive than glucose or carbohydrates, thus remarkably contributing to high production costs of PHA bioplastics. Raistonia eutropha is a representative PHA-producer and can accumulate PHA polymers up to 70-80 wt % of cell mass. This non-sporulating, gram-negative aerobe grows on simple carbon sources and mineral salts. In the presence of glucose and propionic acid or valeric acid, R. eutropha synthesizes P3HB3HV co-polymers with a 3HV content of 5 to 25 mol %. Because of relatively inefficient incorporation of the organic acids into PHA backbone, a high acid concentration is often maintained in the culture medium, resulting in a high toxicity to the cells.
In U.S. Pat. No. 5,364,778 a possible solution to the above problem is disclosed, wherein a microbiological process for producing copolymers comprising HB and HV monomer units using a PHB accumulating bacterium which is not capable of significant growth when cultivated under non growth limiting conditions on a substrate consisting essentially of a HV component. Therefore, at least part of the cultivation is conducted under growth limitation conditions, i.e. under conditions wherein an essential requirement for growth but not copolymer accumulation would be limited. Under such growth limitation conditions the tendency of the bacterium to produce and accumulate PHB homopolymer would be avoided, and the production and accumulation of HV containing copolymer would be induced.